Mycobacterium tuberculosis is a pathogen that evades the host immune system by living within alveolar and peripheral macrophages. Host evasion is partially accomplished by M. tb coating itself with complement fragment 3d (C3d), which directs it for phagocytosis by the host macrophage and inhibits the full Complement response. Because C3d is generated only during specific types of inflammatory events and binds its target rapidly, C3d serves as an excellent biomarker for imaging infections and other specific inflammatory events.
The complement system is an important arm of the innate immune system, providing critical protection against invasive pathogens (Ricklin et al., 2001)) and contributing to the pathogenesis of numerous autoimmune and inflammatory diseases (Walport, 2001). During the course of complement activation, the C3 protein undergoes proteolytic cleavage at several different sites (FIG. 1). The cleavage fragments are fixed to nearby tissues through a covalent linkage originating from the thioester site on C3 with hydroxyl or primary amine groups on acceptor surfaces (3-5). Thus, the deposition of C3 fragments on tissue surfaces constitutes a durable signal of tissue inflammation. For this reason, tissue-bound C3 fragments are commonly used clinically and experimentally as biomarkers of immune activation. Renal biopsies from patients with glomerulonephritis, for example, are routinely immunostained for C3 fragments, and the detection of glomerular C3 fragments serves as a sensitive and robust indicator of disease activity (Schulze et al., 1993). C3 deposition has also been recognized to occur in all stages of age-related macular degeneration (Hageman et al., 2001).
Because tissue-bound C3 fragments are associated with local inflammation, they also have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents in several tissues, including the kidneys, the heart, the brain, and the eyes (Atkinson et al., 2005; Serkova et al., 2010; Sargsyan et al., 2012; Rohrer et al., 2009; Rohrer et al., 2012). These targeted agents have employed recombinant forms of complement receptor 2 (CR2), a protein that can discriminate between intact C3 in the plasma and tissue-bound C3 fragments. The rationale for this approach is that systemically administered agents can be delivered to sites of inflammation through their affinity with the iC3b and C3d fragments. By directing therapeutic agents to molecular targets, one can achieve a high degree of local activity with the drug while minimizing its systemic side effects (Webb, 2011). Previous studies also have used a CR2-targeted contrast agent to detect tissue-bound C3 fragments and renal disease activity by MRI (Serkova et al., 2010; Sargsyan et al., 2012). Although specific for the cleaved forms of C3, CR2-targeted agents probably bind these fragments with a relatively low affinity (reported values range from 1 to 10 μM at physiologic ionic strength) (Guthridge et al., 2001; Isenman et al., 2010; Dempsey et al., 1996). Higher-affinity targeting vectors for epitopes on the cleaved forms of C3 could potentially deliver therapeutic and diagnostic agents to sites of inflammation with even greater efficiency, durability, and specificity.
Informative monoclonal antibodies (mAbs) against tissue-bound C3 fragments have many biomedical applications. They could be used as in vivo delivery vehicles for new therapeutic and diagnostic agents. They also could potentially modulate the biologic functions of the C3 fragments. Such antibodies also could be useful for identifying specific C3 fragments (e.g., C3b, iC3b, C3dg, and C3d) and quantifying their relative abundance. There are, however, several barriers to the generation of such antibodies by standard methods. Like CR2, the antibodies must recognize epitopes of cleaved C3 that are not exposed on intact C3 (which circulates at a concentration of 1 to 2 mg/ml). This is feasible, however, since internal regions of C3d (and also iC3b and C3dg) are exposed by conformational changes in C3 during its activation and subsequent proteolytic processing of its fragments (Janssen et al., 2006). Another difficulty is that standard methods for generating and cloning hybridomas may expose the hybridoma cells to C3 and C3 fragments in serum-containing media, or to C3 synthesized by cells, such as macrophages, that are used in the cultures. C3 and C3 fragments in the media could mask positive hybridoma clones or affect the growth of such clones through engagement of the B cell receptors.